Search

41 p.-7 fig., ICAP-1 regulates β1 integrin activation and cell adhesion. Here we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single positive (SP) CD8+ cell generation, thus unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T and B cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B cell numbers., This work was supported by grants SAF2017-85146-R and PID2020-116291RB-I00 from the Ministerio de Ciencia e Innovación (MICINN) to J.T, PID2019-105623RB-I00 from MICINN to M.L.T,BFU2013-48828-P from MICINN to Y.R.C., ERC Synergy Grant (2018) to R.F., RTI2018-095497-B-I00 from MICINN to A.H, and RTI2018-093938-B-I100 from MICINN and (RD16/0011/0002, TERCEL) from Instituto de Salud Carlos III to AGZ.

252 p.-91 fig.-7 tab., [EN]The integrin α4β1 plays important roles during lymphocyte migration to sites of inflammation and at distinct steps in hematopoiesis. The activity of this integrin is regulated by intracellular proteins which associate with the β1 subunit cytoplasmic domain. While much is known on positive regulators such as talin and kindlin, less is known about those which negatively regulate integrin activation, such as ICAP-1. As α4β1 is expressed on lymphocytes, we have studied the effects of the lack of ICAP-1 on α4β1-dependent lymphocyte adhesion and on immune cell differentiation in thymus, spleen and bone marrow (BM). α4β1 also mediates the interaction between multiple myeloma (MM) cells and the BM microvasculature, as well as with BM stromal cells. This interaction favours MM cell proliferation, survival and resistance to chemotherapy, leading to BM colonization and disease progression. Given the potential role of ICAP-1 as negative regulator of β1 integrin activation, it is important to study its role on α4β1-dependent MM cell adhesion. Bortezomib (BTZ) is a proteasome inhibitor widely used in the treatment against MM. Unfortunately, many patients develop resistance to BTZ, and therefore is crucial to investigate the mechanisms that the MM cells use to bypass this inhibitor, including the possible relationships between BTZ and α4β1. In the first objective of this Doctoral Thesis we have characterized the ICAP-1 implication in α4β1-mediated lymphocyte adhesion, and in the development and maturation of cells of the immune system. In addition, we have analyzed the α4β1-dependent MM cell adhesion to the BM microvasculature., To address the first part of this objective, we used mice lacking ICAP-1. CD3 and -CD19 splenic cells from ICAP-1 mice displayed higher adhesion levels to α4β1 ligands than ICAP-1 cells. ICAP-1-deficient mice showed a specific reduction of TCRαβ hiCD8 CD69 cells in comparison with control mice, suggesting an involvement of ICAP-1 in thymocyte positive selection. The decreased CD8 T cell frequency was also evident in spleen and lymph nodes from ICAP-1 mice. Contrary to spleen cells, there were no differences in adhesion to CS-1/FN between thymocytes from ICAP-1 and ICAP-1 mice. Interestingly, we observed a preferential distribution of ICAP-1 in the nucleus of control thymocytes, suggesting that ICAP-1 function in these cells might be more relevant in the nucleus than in the cytosol. Analyzing regulatory elements of the thymocyte development program, we found that lack of ICAP-1 caused a specific reduction in the expression of Runx3, an important transcription factor controlling the generation of CD8 cells in thymus. We did not detect alterations in stem and progenitor cells frequencies in BM from ICAP-1 mice relative to control counterparts, and B lymphopoiesis was neither affected. Of note, we observed a reduction of marginal zone B cells and an increase in follicular B cells in spleen from ICAP-1 mice comparing to control mice. These data suggest that ICAP-1 might have a role as modulator of B cell trafficking in spleen, thus regulating B cell maturation. In the second part of this objective we demonstrated that ICAP-1 is expressed in MM bone marrow primary samples, and that silencing ICAP-1 in MM cell lines led to enhanced α4β1-dependent adhesion together with increased integrin activation. Furthermore, intravital microscopy assays revealed that ICAP-1 knockdown led to upregulated MM cell adhesion to the BM microvasculature. The second objective consisted in the characterization of functional relationships between MM cell resistance to BTZ and the expression and function of the α4β1 integrin. To this purpose we have used two MM cell lines resistant to BTZ. We found that the expression of α4β1 in these BTZ-resistant cells was higher than that of the original parental cells, which resulted in increased α4β1-dependent cell adhesion and enhanced cell infiltration in the BM of immunodeficient mice, which caused disease progression. A partial involvement of NF-κB and FOXO3a in enhanced α4 expression in the resistant cells was found. Upregulation of α4β1 expression in BTZ-resistant cells was not a general effect, as it was not observed in BTZ-resistant cells which served as models for acute lymphoblastic leukemia T and B cell lymphoma., [ES]La integrina α4β1 desempeña importantes funciones durante la migración linfocitaria hacia sitios de inflamación y en diferentes etapas de la hematopoyesis. La actividad de esta integrina está regulada por proteínas intracelulares que se asocian al dominio citoplasmático de la subunidad β1. Mientras que se han realizado numerosos estudios acerca de los reguladores positivos de las integrinas tales como talina y kindlin, aquellas proteínas que regulan negativamente su activación han sido menos caracterizadas, como es el caso de ICAP-1. Puesto que α4β1 se expresa en linfocitos, hemos estudiado el efecto de la falta de ICAP-1 en la adhesión dependiente de α4β1 en estas células, así como en la diferenciación de células del sistema inmunitario en el timo, bazo y médula ósea (MO). Otra de las funciones conocidas de α4β1 es su participación en la interacción entre células de mieloma múltiple (MM) y la microvasculatura de la MO y células del estroma medular. Esta interacción favorece la proliferación, supervivencia y resistencia a quimioterapia de las células de MM, promoviendo la colonización de la MO y la progresión de la enfermedad. Debido a que ICAP-1 se postula como regulador negativo de la activación de integrinas β1, es importante estudiar su papel en la adhesión de células de MM dependiente de α4β1. El Bortezomib (BTZ) es un inhibidor del proteasoma ampliamente utilizado en el tratamiento frente al MM. Desafortunadamente, muchos pacientes acaban desarrollando resistencia al BTZ, por lo que es crucial investigar los mecanismos que utilizan las células de MM para sobrevivir al tratamiento con este agente, incluyendo una posible relación entre el BTZ y la integrina α4β1. En el primer objetivo de esta Tesis Doctoral hemos caracterizado la implicación de ICAP-1 en la adhesión linfocitaria dependiente de α4β1, y en el desarrollo y maduración de células del sistema inmunitario. A su vez, hemos analizado la regulación por ICAP-1 de la adhesión de células de MM a la microvasculatura de la MO a través de α4β1., Para abordar la primera parte de este objetivo hemos utilizado ratones deficientes en ICAP-1. Esplenocitos CD3 y CD19 procedentes de ratones ICAP-1 mostraron niveles mayores de adhesión a ligandos de α4β1 que las células ICAP-1 . Detectamos una reducción hi específica de la población de células TCRαβ CD8 CD69 en muestras de timos de ratones ICAP-1 en comparación con muestras ICAP-1 , sugiriendo un posible papel de ICAP-1 en la selección positiva de los timocitos. El descenso de la frecuencia de células T CD8 también fue notorio en bazo y nódulos linfáticos de ratones ICAP-1 . Contrariamente a lo observado para los esplenocitos, no se encontraron diferencias en los niveles de adhesión a CS-1/FN entre timocitos ICAP-1 y ICAP-1 . Notablemente, detectamos una distribución preferencial de ICAP-1 en el núcleo de los timocitos, sugiriendo que la función desempeñada por esta proteína en estas células podría ser más relevante en el núcleo que en el citoplasma. Analizamos elementos reguladores del programa de desarrollo por el que los timocitos se determinan hacia los distintos linajes de células T, y encontramos que la falta de ICAP-1 causaba una reducción específica de la expresión de Runx3, un factor de transcripción crítico para la generación de células CD8 en el timo. No detectamos alteraciones en la frecuencia de células madre y progenitoras presentes en la MO de ratones ICAP-1 , ni en la linfopoyesis de células B. El análisis de los bazos de ratones ICAP-1 mostró una reducción de las células B de la zona marginal (MZ) y un incremento de las células B foliculares (FO), en comparación con los bazos de ratones control. Estos datos sugieren la posibilidad de que ICAP-1 actúe como modulador del tráfico de células B en bazo, por lo que pudiera regular la maduración de las células B. En la segunda parte de este objetivo silenciamos ICAP-1 en líneas celulares de MM, lo que supuso un incremento de la adhesión dependiente de α4β1 junto con una mayor activación de la integrina. Ensayos de microscopía intravital revelaron que la reducción de la expresión de ICAP-1 correlacionaba con un aumento de la adhesión de las células de MM a la microvasculatura de la MO. El segundo objetivo de esta Tesis consistió en la caracterización de relaciones funcionales entre la resistencia de células de MM a BTZ y la expresión y función de α4β1. Para desarrollar este trabajo hemos utilizado líneas celulares de MM resistentes a BTZ. Hemos observado que la expresión de α4β1 en estas células resistentes era mayor que la de las células parentales, lo que se tradujo en un aumento en los niveles de adhesión mediada por esta integrina y en una mayor infiltración de la MO de ratones inmunodeficientes, contribuyendo al progreso de la enfermedad. Los factores de transcripción NF-κB y FOXO3a están involucrados en el aumento de la expresión de α4 en las poblaciones celulares de MM resistentes a BTZ. El incremento de expresión de α4β1 en las células de MM resistentes a BTZ no resultó ser un efecto extensible a otras neoplasias hematológicas, ya que no se observó en células resistentes a BTZ utilizadas como modelos de leucemia linfoblástica aguda de células T y linfoma de células B del manto. Nuestros resultados indican que ICAP-1 está implicado en el desarrollo de células T CD8 en el timo, regulando la expresión de Runx3 y modulando la selección positiva de los timocitos. ICAP-1 controla negativamente la adhesión dependiente de α4β1 de células T y B del bazo, y regula el tráfico de células B en este órgano afectando a la frecuencia de células MZ y FO. Demostramos asimismo que ICAP-1 regula negativamente la adhesión de células de MM mediada por α4β1. Las células de MM resistentes a BTZ muestran mayor expresión de α4β1 en comparación a las células parentales, lo que causa un aumento de la adhesión mediada por esta integrina, así como una mayor infiltración de la MO y progresión del mieloma. Proponemos a NF-κB y FOXO3a como reguladores del aumento de la expresión de α4 en células de MM resistentes a BTZ., Financiación del proyecto (SAF2014-53059-R) del MINECO

ICAP-1 prevents recruitment of kindlin-2 to β1 integrin to control dynamics of fibrillar adhesion sites, fibronectin deposition, and osteoblast mineralization during bone formation., The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.

International audience; The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix interactions through the recruitment of accessory molecules. One of them, the integrin cytoplasmic domain-associated protein-1 (ICAP-1; also known as ITGB1BP1), specifically interacts with the cytoplasmic domain of the beta(1) integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. We report an unexpected role of ICAP-1 in osteoblast function during bone development. Icap-1-deficient mice suffer from reduced osteoblast proliferation and delayed bone mineralization, resulting in the retarded formation of bone sutures. In vitro studies reveal that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates, probably owing to an increase in beta(1) integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state.

Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1 alpha (integrin cytoplasmic domain-associated protein-1 alpha) binds to the cytoplasmic domain of the beta(1A) integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with beta(1A) integrin, ICAP-1 alpha is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1 alpha induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1 alpha to inhibit the association of beta(1A) integrin with talin, which is crucial for the assembly of these structures. ICAP-1 alpha-mediated dispersion of beta(1A) integrins is not observed with beta(1D) integrins that do not bind ICAP. This strongly suggests that ICAP-1 alpha action depends on a direct interaction between ICAP-1 alpha and the cytoplasmic domain of the beta(1) chains. Altogether, these results suggest that ICAP-1 alpha plays a key role in cell adhesion by acting as a negative regulator of beta(1) integrin avidity.

International audience; The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix interactions through the recruitment of accessory molecules. One of them, the integrin cytoplasmic domain-associated protein-1 (ICAP-1; also known as ITGB1BP1), specifically interacts with the cytoplasmic domain of the beta(1) integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. We report an unexpected role of ICAP-1 in osteoblast function during bone development. Icap-1-deficient mice suffer from reduced osteoblast proliferation and delayed bone mineralization, resulting in the retarded formation of bone sutures. In vitro studies reveal that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates, probably owing to an increase in beta(1) integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state.

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the β1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of β1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a β1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the β1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor–induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.